Recent studies have identified Cytolysin-Mediated Translocation (CMT), a novel translocation pathway in the Gram-positive pathogen Streptococcus pyogenes that utilizes the pore-forming cytolysin Streptolysin O (SLO) to translocate the S. pyogenes NAD-glycohydrolase (SPN) into the host cell cytosol. However, how SLO functions to translocate SPN and how SPN contributes to pathogenesis are unknown. A central question is how the CMT pathway generates a polarized distribution of the effector molecule where the majority of the SPN exported from the bacterium by the non-specialized general secretory (Sec) pathway is destined for the host cell cytosol, and is not released into the extracellular milieu. Valuable insight has come from our recent studies that have defined a domain in SLO that is essential for effector translocation, but is dispensable for formation of transmembrane pores. We have also shown that interactions between SPN and the soluble SLO monomer are not readily detectable, that CMT has the ability to discriminate between Sec substrates, routing SPN into the host cell cytosol, while directing others into the extracellular spaces, and that production of SPN requires a putative chaperone protein we have named IPS (Immunity Factor for SPN). These studies establish several important points: 1.) that pore formation by itself is not sufficient for CMT; 2.) that CMT is driven by specific protein-protein interactions involving SLO; 3.) that these interactions likely involve multimeric and not monomeric SLO; and 4.) that these interactions are either with SPN itself and/or the components of an export channel. This Project will explore the nature of these interactions and will examine the role of SPN's enzymatic activities in pathogenesis. Understanding these issues will be important for the identification of additional effector proteins and for evaluating whether CMT contributes to the virulence of other Gram-positive pathogens.